1. Field of the Invention
This invention resides in the field of affinity chromatography, and addresses concerns arising from the labile character of ligands coupled to a support as the stationary phase.
2. Description of the Prior Art
Affinity chromatography is widely used for separating and detecting components in biological samples and for the isolation or purification of biological species or recombinant species from clinical samples, cell growth cultures, or any medium in which the species are produced or can be extracted. Affinity chromatography is commonly performed by passing a liquid medium containing the species of interest through a column or membrane to which a ligand is bound as a stationary phase, the ligand being one to which the species of interest binds by an affinity-type interaction. Affinity chromatography that is used for isolation and purification is also termed “affinity extraction,” and the species-ligand interaction in this type of extraction is one that occurs with sufficient specificity to differentiate between the species of interest and other species in the source liquid. Affinity extraction techniques include immunoextraction in which the ligands are antibodies; protein-protein extractions using such ligands as wheat germ agglutinin, concanavalin A, protein A, and protein G; and interactions involving non-protein species such as heparin or nucleic acids. Once the species of interest is immobilized by the bound ligand as a result of the affinity interaction, ligand and its support are washed to remove unbound species and the bound species is then released from the ligand. Release is effected by an appropriate change in conditions such as a change in pH or the introduction of a detergent, chaotrope, salt, competitive binding species, or any agent that will overcome or lessen the binding affinity of the species to the ligand. The types of changes that will be effective in releasing the bound species in particular systems are well known in the art of affinity chromatography.
The ligand is typically a protein or other affinity-binding species that is coupled by covalent bonding to a solid support to form the stationary phase, the support often having been activated to facilitate the covalent bonding. Activation commonly involves the placement of a reactive group, one example of which is an epoxide group, on the support surface. The linkage between the ligand and the support is typically labile, however, leaving the ligand prone to dissociation from the support as the sample and other liquids pass through the medium. In addition to dissociation due to a simple shift in equilibrium, dissociation can also occur as the result of enzymatic or chemical degradation of the ligand itself. Proteases in the process stream can cause proteolysis of protein affinity ligands, for example, and endo- and exo-nucleases can cause cleavage of nucleic acid ligands. The amount of ligand that is leached as a result of this dissociation may be small compared to the amount of ligand remaining on the support, but even a small amount of leached ligand can seriously contaminate the otherwise purified species eluted from the medium. When a therapeutic agent that is either biologically derived or produced by recombinant chemistry is contaminated with a leached affinity ligand, the leached ligand can recombine with the agent and thereby impede the effectiveness of the agent, or bind to, or impede the functions of, other species or tissues in the patient's body, such as membranes, cell walls, or enzymes, causing harm. Concanavalin A, for example, is an affinity ligand that is used for purifying lysosomal enzyme preparations, but is known to leach from affinity columns and contaminate the enzyme preparations, particularly by activating T cells in the patient to whom the enzyme preparation is administered. To eliminate these types of contamination, the leached ligands must be removed, and this is typically performed by separations downstream of the affinity column or membrane. This adds cost and time to the preparation.